Identifying genetic variation in affected tissues

ABSTRACT

Methods for the determination of tissue-specific genetic variation are provided. For example, in certain aspects methods for using iPS cell-derived specific cell types for differential molecular analysis of tissue-specific genetic variation are described.

The present application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 61/237,908, filed Aug. 28, 2009, the entirecontents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of moleculardiagnosis. More particularly, it concerns the use of induced pluripotentstem cells (iPS cells) in determination and characterization of geneticvariation.

2. Description of Related Art

Pathology caused by defects in human genes is usually highlytissue-specific. In heritable diseases, this suggests that specificspatiotemporal functions of the implicated genes are disrupted due togerm-line mutations. Although it has been shown that disease genesgenerally tend to be expressed in a limited number of tissues, it isstill unclear in many cases how the tissue-specific expression patternsor genetic structure of disease genes correlate with their pathologicalor abnormal manifestations, and it remains difficult to determine orcharacterize the abnormal genes or genetic structure in certain tissueor cell types.

In a broader aspect, understanding genetic variation is a key goal ofhuman genetics, encompassing disease susceptibility, variable responseto drugs and ultimately treatment and public health.

The human body is assembled from more than 200 cell types present in avariety of tissue types. For identifying unknown genetic defects ordetermining genetic variation, specific cell types need to be isolatedand characterized. However, some cell types may be hard to obtain from ahuman subject in vivo, such as the highly specialized retinal pigmentepithelium (RPE) cells. Thus, there remains a need to develop moreconvenient methods to provide specific cell types to analyze thetissue-specific genetic variation.

SUMMARY OF THE INVENTION

The present invention overcomes a major deficiency in the art byproviding tissue-specific genetic variation diagnosis methods relevantto the use of iPS cells, specifically, by providing specificdisease-relevant cell types which may be challenging to isolate orobtain. In a first embodiment, there is provided a method fordetermining the presence of a tissue-specific genetic variation in atest subject relative to a selected control genetic structure,comprising: a) obtaining genetic material of a differentiated cell of aselected tissue, the cell having been prepared by differentiating aninduced pluripotent stem (iPS) cells obtained by reprogramming a somaticcell of the test subject; and b) testing the genetic material of thedifferentiated cell to compare one or more genetic structure of thegenetic material with one or more control genetic structures todetermine the presence of such a genetic variation in cells of thetissue of the test subject. In certain embodiments, the selected tissuemay be retina, neural tissue, or cardiac muscle tissue.

In certain aspects, the subject may have or be suspected of having agenetic abnormality or genetic disease. The genetic disease may be atissue-specific disease with genetic defects difficult to characterize,such as a retinal disease. For retina disease characterization, specificretina cell types, such as a neural retina cell or a retina pigmentepithelium (RPE) cell, could be provided by certain aspects of thepresent methods, specifically by differentiating iPS cells reprogrammedfrom a somatic cell derived from the test subject.

The genetic material of the differentiated cell, which may comprisenucleotides such as RNA or DNA, could be tested by various ways known inthe art, such as nucleotide sequencing, e.g., RNA or DNA sequencing, ormicroarray analysis. The genetic structure may be primary nucleotidesequence, secondary structure, epigenetic structure, chromosomestructure and the like. In further aspects of the invention, the geneticstructure of the differentiated cell may be represented by an expressionprofile, which may reflect the functional consequences oftranscriptional control regions within DNA sequences, which are notapparent from simple nucleotides sequencing alone.

Certain aspects of the present methods may be used to identify geneticvariation, which may be variation of one or more coding sequences, orvariation of one or more non-coding sequences, such as gene regulatoryelements. The genetic variation determined by those steps above could beany types of mutations, such as deletion, insertion, mismatches,translocation, duplication, inversion, loss of heterozygosity; orpolymorphism, such as single-nucleotide polymorphism (SNP); orrepresented by differential expression. Also the genetic variation canbe a difference in transcript abundance resulting from differences inthe functioning of expression control elements in the patient tissues.

The present methods may be particularly useful for determining thepresence of genetic variation such as polymorphisms in non-codingregions, like introns or regulatory regions including, but are notlimited to, promoters, enhancers, silencers, responsive elements, orregulatory sequences on RNA, such as 5′- or 3′-UTR (untranslatedregions), or any uncharacterized regulatory elements. The geneticvariation may also comprise variation in non-coding RNA, such asmicroRNA, or epigenetic variation, such as variation in modification ofthe genetic structure, e.g., methylation.

To determine the presence of such a genetic variation, a selectedcontrol gene structure may be used, such as one without the selectedgenetic variation. In a certain aspect, the control gene structure maybe comprised in genetic material from a normal tissue, preferentiallysuch a normal tissue may be from a control subject such as sibling orother family member of the test subject. The control subject may nothave the genetic variation, especially in the tissue type of theselected tissue of the test subject. In a further aspect, the controlgene structure may be comprised in genetic material, wherein the geneticmaterial is from a selected normal cell prepared by differentiating aniPS cell obtained by reprogramming a normal somatic cell of a controlsubject, such as a subject who does not have the genetic variation.Preferably, the selected normal cell or the normal tissue may be of thesame tissue type as the selected tissue of the test subject forcomparison of tissue-specific genetic structure.

In certain aspects, the control subject may be a family member relatedto the test subject.

Embodiments discussed in the context of methods and/or compositions ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the terms “encode” or “encoding” with reference to anucleic acid are used to make the invention readily understandable bythe skilled artisan; however, these terms may be used interchangeablywith “comprise” or “comprising” respectively.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. Introduction

The present disclosure, according to certain embodiments, is generallydirected to methods for determining tissue-specific genetic variation bytesting genetic material of specific cell types that may be derived frominduced pluripotent stem cells. The genetic material may comprise anynuclear (chromosomal, DNA, RNA, etc.) and cytoplasmic (e.g.,mitochondrial, proteins) material that could play a fundamental role indetermining the nature of cell substances, cell structures, and/or celleffects. The genetic variation to be determined may include, forexample, genetic defects or genetic polymorphism.

Genetic defects, which may result in a disease or disorder that isinherited genetically, comprise abnormalities in genetic structures,such as an absent or defective gene or a chromosomal aberration. Thosegenetic defects determined by certain aspects of the present inventioncould be applied for genetic disease diagnosis or even therapy.

Genetic polymorphism in specific tissue or cell types may also bedetermined by certain aspects of the present methods. For example,genetic polymorphism refers to the occurrence of more than one allele orgenetic marker at the same locus, with the least frequent allele ormarker occurring more frequently than can be accounted for by mutationalone. Genetic polymorphisms may include Single Nucleotide Polymorphisms(SNPs); Restriction Fragment Length Polymorphisms (RFLPs); Copy NumberPolymorphisms; polymorphisms in transcription or expression, such asvariation in RNA levels, timing or tissue distribution; polymorphisms innon-coding regions, including introns and regulatory elements, such aspromoters, silencers, enhancers, responsive elements, transcriptionfactors, untranslated regions, or non-coding RNA; trinucleotide repeatpolymorphisms, for example, the number, dynamics and/or distribution oftrinucleotides, such as CAG repeats; polymorphisms in epigenetic status,for example, methylation states; differential sensitivity to metabolismchanges, growth factors (e.g., insulin), chemical or pharmaceuticalagents, therapeutic procedures or treatments, or the like;susceptibility to a disease or disorder; variation in drug response,metabolism or toxicity, etc. For example, determination of geneticpolymorphisms may be helpful for disease diagnosis or prognosis fortheir correlation to the occurrence of genetic defects or carrierstatus.

In certain aspects, genetic material testing may comprise determiningpolymorphism of RNA expression or sequence of the specific cell types.The cell-specific RNA characterization may be advantageous in yieldinginformation that even complete DNA genome sequencing or complete whatproteomics sequencing cannot yield. For example, as the functionalconsequences of variations in DNA control regions are not yet wellunderstood, examining the RNA in the target tissue or cell types (wholetranscriptome analysis) may be able to identify functional variations inthe regulatory elements or regions that could not be identified byhaving an entire genome sequence sequenced and may provide informationunavailable by whole genome sequencing. Particularly, gene expressionlevel (e.g., transcription) is quantitative measure that is directlylinked to genetic variation and can usually represent polymorphisms inregulatory elements. If a polymorphism or mutation in the regulatoryelements is related to a tissue-specific disease or abnormality, it maybe represented by changes in the RNA expression levels, localization,timing, or relative levels of spliced variants in the relevant tissue.RNA sequencing of the specific cell types may also reveal the specificvariation much faster than DNA sequencing, as the inventors specificallyexamine the genetic material related to that cell type without requiringwhole genome DNA sequences. This analysis would be best done inside-by-side comparison to unaffected siblings, to have the greatestchance of identifying the transcript differences associated with thedisease condition.

The present invention could be a prelude to treating the tissue-specificdisease by transplantation of differentiated cells derived fromgenetically corrected iPS cells, or a diagnostic of tissue- orcell-specific genetic variation alone. Additionally, certain aspects ofthis invention dramatically reduce the complexity of the identificationof the genetic cause or the genetic marker for uncharacterized geneticdisorders, since the inventors may start with the polymorphismsrepresented by difference between the control and test subjectexpression profiles rather than the whole genome or transcriptome.

II. Genetic Variation and Genetic Disease

Certain aspects provide methods to determine the presence of geneticvariation, which may provide diagnosis of genetic diseases orabnormality. Genetic variation may play a key role in shaping phenotypicdiversity amongst individuals, wherein the underlying mechanisms mayinclude polymorphisms or abnormalities that alter protein codingsequences or changes in regulatory sequences that affect the function orexpression of a gene or related gene networks. Genetic variation mayresult from the presence of different genotypes in the population and beassociated with, render susceptibility to, or be the underlying causefor genetic disease or abnormality.

Genetic diseases may be caused by germ-line mutations that, despitetissue-wide presence, often lead to tissue-specific pathology, orabnormality in operation of regulatory elements, which may also have atissue-specific phenotype.

A. Genetic Variation

Genetic variation may influence gene expression in specific tissue orcell types. In certain aspects of the present methods, by associatingtissue or cell-specific expression with genetic structure, geneticvariation could be identified or determined. For example, geneticvariation may include polymorphisms such as single nucleotidepolymorphisms (SNPs), which are DNA sequence variations occurring when asingle nucleotide—A, T, C, or G—in the genome (or other shared sequence)differs between members of a species (or between paired chromosomes inan individual). Analysis of diploid sequences has also shown thatnon-SNP variation accounts for much more human genetic variation thansingle nucleotide diversity. This non-SNP variation includes copy numbervariation and may result from deletions, inversions, insertions andduplications. It is estimated that approximately 0.4% of the genomes ofunrelated people typically differ with respect to copy number.

Some of the polymorphisms may be in non-coding regions of the genomewhile some may reside in the coding regions, for example, polymorphismsin regulatory regions or elements, or exonic variants alteringtranscript stability or splicing.

Genetic variation may include genetic regulatory polymorphisms acting incis by changing the protein coding sequence or at the level of RNA:affecting transcription (activation or inhibition through regulatorysites or structure of regulatory elements), mRNA processing, pre-mRNAsplicing, exonic splicing enhancers (ESEs), exon skipping, mRNAstability, mRNA trafficking, or regulatory RNAs. Epigenetic variationmay mimic regulatory polymorphisms to some extent (Johnson et al.,2005).

Polymorphisms in regulatory regions or elements may be one of the keyprimary effects contributing to phenotypic variation in humans and thusimportant for molecular analysis of human phenotypic variation, some ofwhich may be linked to genetic disorders.

For example, there is growing evidence that regulatory polymorphismsplay an important role in the determination of individual susceptibilityto complex disease traits (Knight 2005). Such regulatory polymorphismsinclude, but are not limited to, variation at the Variable Number TandemRepeat (i.e., VNTR) of INS, encoding insulin, in type 1 diabetes;polymorphism of CTLA4, encoding cytotoxic T lymphocyte antigen, inautoimmune disease; polymorphism of Duffy binding protein in malaria;polymorphism of CCR5, encoding chemokine receptor 5, in HIV-1 infection.Genetic variation may also operate at other levels of control of geneexpression and the modulation of splicing at PTPRC, encoding proteintyrosine phosphatase receptor-type C, and of translational efficiency atF12, encoding factor XII.

B. Genetic Disorders

A genetic disease (i.e., genetic disorder) is an illness caused byabnormalities in genes or chromosomes, or in gene regulatory elements.Abnormalities can range from a small mutation in a single gene to theaddition or subtraction of an entire chromosome or set of chromosomes.Some diseases, such as cancer, are due in part to genetic disorders andalso in part caused by environmental factors. Some types of recessivegene disorders confer an advantage in the heterozygous state in certainenvironments. A haploid cell has only one set of chromosomes. A diploidcell has two sets of chromosomes. In human, the somatic cells arediploid, and the gametes are haploid.

The range of genetic diseases is so broad in nature that affectedindividuals can be found in virtually all medical practices. Simply put,genetic blueprint influences health from the moment of conception untildeath. Based on a population study of genetic disorders apparent by theage of 25 years (Baird et al., 1988), about 0.4% of the population havea single gene (Mendelian) disorder, 0.2% have a chromosomal abnormalityand 4.6% have a multifactorial condition. Another 0.1% have an obviousgenetic abnormality of unknown inheritance, and 0.3% have congenitalproblems that are not genetic in nature. A single gene disorder is theresult of a single mutated gene. There are estimated to be over 4000human diseases caused by single gene defects. Single gene disorders canbe passed on to subsequent generations in several ways. Genomicimprinting and uniparental disomy, however, may affect inheritancepatterns. The divisions between recessive and dominant types are not“hard and fast” although the divisions between autosomal and X-linkedtypes are (since the latter types are distinguished purely based on thechromosomal location of the gene). For example, achondroplasia istypically considered a dominant disorder, but children with two genesfor achondroplasia have a severe skeletal disorder that achondroplasicscould be viewed as carriers of. Sickle-cell anemia is also considered arecessive condition, but heterozygous carriers have increased immunityto malaria in early childhood, which could be described as a relateddominant condition.

Only one mutated copy of the gene will be necessary for a person to beaffected by an autosomal dominant disorder. Each affected person usuallyhas one affected parent. There is a 50% chance that a child will inheritthe mutated gene. Conditions that are autosomal dominant often have lowpenetrance, which means that although only one mutated copy is needed, arelatively small proportion of those who inherit that mutation go on todevelop the disease. Examples of this type of disorder are Huntington'sdisease, Neurofibromatosis 1, Marfan Syndrome, Hereditary nonpolyposiscolorectal cancer, and Hereditary multiple exostoses, which is a highlypenetrant autosomal dominant disorder. Birth defects are also calledcongenital anomalies.

Two copies of the gene must be mutated for a person to be affected by anautosomal recessive disorder. An affected person usually has unaffectedparents who each carry a single copy of the mutated gene (and arereferred to as carriers). Two unaffected people who each carry one copyof the mutated gene have a 25% chance with each pregnancy of having achild affected by the disorder. Examples of this type of disorder arecystic fibrosis, sickle-cell disease (also partial sickle-cell disease),Tay-Sachs disease, Niemann-Pick disease, spinal muscular atrophy, andDry (otherwise known as “rice-brand”) earwax.

X-linked dominant disorders are caused by mutations in genes on the Xchromosome. Only a few disorders have this inheritance pattern, with aprime example being X-linked hypophosphatemic rickets. Males and femalesare both affected in these disorders, with males typically being moreseverely affected than females. Some X-linked dominant conditions suchas Rett syndrome, Incontinentia Pigmenti type 2 and Aicardi Syndrome areusually fatal in males either in utero or shortly after birth, and aretherefore predominantly seen in females. Exceptions to this finding areextremely rare cases in which boys with Klinefelter Syndrome (47, XXY)also inherit an X-linked dominant condition and exhibit symptoms moresimilar to those of a female in terms of disease severity. The chance ofpassing on an X-linked dominant disorder differs between men and women.The sons of a man with an X-linked dominant disorder will all beunaffected (since they receive their father's Y chromosome), and hisdaughters will all inherit the condition. A woman with an X-linkeddominant disorder has a 50% chance of having an affected fetus with eachpregnancy, although it should be noted that in cases such asIncontinentia Pigmenti only female offspring are generally viable. Inaddition, although these conditions do not alter fertility per se,individuals with Rett syndrome or Aicardi syndrome rarely reproduce.

X-linked recessive disorders are also caused by mutations in genes onthe X chromosome. Males are more frequently affected than females, andthe chance of passing on the disorder differs between men and women. Thesons of a man with an X-linked recessive disorder will not be affected,and his daughters will carry one copy of the mutated gene. A woman whois a carrier of an X-linked recessive disorder (XRXr) has a 50% chanceof having sons who are affected and a 50% chance of having daughters whocarry one copy of the mutated gene and are therefore carriers. Examplesof this type of disorder are Hemophilia A, Duchenne muscular dystrophy,red-green color blindness, Muscular dystrophy and Androgenetic alopecia.

Y-linked disorders are caused by mutations on the Y chromosome. Becausemales inherit a Y chromosome from their fathers, every son of anaffected father will be affected. Because females inherit an Xchromosome from their fathers, female offspring of affected fathers arenever affected. Since the Y chromosome is relatively small and containsvery few genes, there are relatively few Y-linked disorders. Often thesymptoms include infertility, which may be circumvented with the help ofsome fertility treatments. Examples are Male Infertility andhypertrichosis pinnae.

Genetic disorders may also be complex, multifactorial or polygenic, thismeans that they are likely associated with the effects of multiple genesin combination with lifestyle and environmental factors. Multifactoraldisorders include heart disease and diabetes. Although complex disordersoften cluster in families, they do not have a clear-cut pattern ofinheritance. This makes it difficult to determine a person's risk ofinheriting or passing on these disorders. Complex disorders are alsodifficult to study and treat because the specific factors that causemost of these disorders have not yet been identified.

C. Consideration of Genetic Disorders or Abnormalities

The same considerations that go into adding a possible genetic disorderto a neonate's or child's differential diagnosis apply to adults. Isthis disorder genetic or acquired? How significant is the geneticcomponent versus the environmental component? What is the pattern ofinheritance? Is there potential for other members of the patient'sfamily to be affected presently or in the future? How can the diagnosisbe confirmed? There are a few clues that will suggest a geneticdiagnosis. The family history might point to a genetic disorder. A greatmany disorders of adult onset are dominant in nature. That is, amutation in only one of a gene pair is necessary to produce a geneticproblem. (This contrasts with recessive inheritance in which both genesof a pair must have a mutation.) Dominant genetic disorders are verylikely to produce a family history, whereas recessive disorders are not.Unfortunately, some dominant conditions have a high new mutation rate,so your patient might well be the first case in the family.Alternatively, a positive family history may not have previously beenappreciated, perhaps because of the small size of the family ordispersion of family members. Family members who carried a gene mutationmay have died before they developed signs and symptoms, or a geneticdiagnosis may have never previously been entertained despite theexistence of affected relatives.

Pedigree analysis may be complicated by the phenomena of incompletepenetrance and variable expressivity (Harper, 1998). Incompletepenetrance means that some individuals may carry a genetic mutation butnot express any signs and symptoms. Variable expressivity means thateven though all affected members of the family have the same mutation,they may not have identical manifestations. Age of onset and severity ofthe condition may vary considerably.

When a disease occurs at a much younger age than one would normallyexpect, it suggests a possible genetic predisposition. For example,breast cancer in a 25-year-old is very unusual (Langston et al., 1996).Paired with a history of breast and ovarian cancer in close relatives,this is virtually diagnostic of a mutation to a BRCA gene (Haber, 1999).

Physicians should also consider a genetic cause in three additionalcircumstances: cases with multisystem involvement (e.g., the deafnessand nephritis associated with Alport's syndrome (Flinter, 1997)), with amultifocal presentation (e.g., the multiplicity of polyps throughout thecolon diagnostic of familial adenomatous polyposis (Midgley and Kerr,1999)) or with an unusual combination of events (e.g., early onsetosteoporosis and conductive hearing loss may be indicative ofosteogenesis imperfecta (Byers and Steiner, 1992)).

D. Genetic Eye Disorders

In particular, the present methods may be used to determine geneticvariation or identify novel genetic defects in specific retina cellstypes differentiated from iPS cells, which may be derived from a patienthaving or suspected of having a genetic eye disorder, such as retinitispigmentosa.

Retinitis pigmentosa (RP) is a group of genetic eye disorders. In theprogression of symptoms for RP, night blindness generally precedestunnel vision by years or even decades. Many people with RP do notbecome legally blind until their 40s or 50s and retain some sight alltheir life. Others go completely blind from RP, in some cases as earlyas childhood. Progression of RP is different in each case.

RP is a type of progressive retinal dystrophy, a group of inheriteddisorders in which abnormalities of the photoreceptors (rods and cones)or the retinal pigment epithelium (RPE) of the retina lead toprogressive visual loss. Affected individuals first experience defectivedark adaptation or nyctalopia (night blindness), followed by reductionof the peripheral visual field (known as tunnel vision) and, sometimes,loss of central vision late in the course of the disease.

The diagnosis of retinitis pigmentosa relies upon documentation ofprogressive loss in photoreceptor function by electroretinography (ERG)and visual field testing. The mode of inheritance of RP is determined byfamily history. At least 35 different genes or loci are known to cause“nonsyndromic RP” (RP that is not the result of another disease or partof a wider syndrome).

There are multiple genes that, when mutated, can cause the Retinitispigmentosa phenotype. In 1989, a mutation of the gene for rhodopsin, apigment that plays an essential part in the visual transduction cascadeenabling vision in low-light conditions, was identified. Since then,more than 100 mutations have been found in this gene, accounting for 15%of all types of retinal degeneration. Most of those mutations aremissense mutations and inherited mostly in a dominant manner. Therhodopsin gene encodes a principal protein of photoreceptor outersegments. Studies show that mutations in this gene are responsible forapproximately 25% of autosomal dominant forms of RP.

Mutations in four pre-mRNA splicing factors are known to cause autosomaldominant retinitis pigmentosa. These are PRPF3, PRPF8, PRPF31 and PAP1.These factors are ubiquitously expressed and it is still a puzzle as towhy defects in a ubiquitous factor should only cause disease in theretina.

Up to 150 mutations have been reported to date in the opsin geneassociated with the RP since the Pro23His mutation in the intradiscaldomain of the protein was first reported in 1990. These mutations arefound throughout the opsin gene and are distributed along the threedomains of the protein (the intradiscal, transmembrane, and cytoplasmicdomains). One of the main biochemical causes of RP in the case ofrhodopsin mutations is protein misfolding, and molecular chaperones havealso been involved in RP. It was found that the mutation of codon 23 inthe rhodopsin gene, in which proline is changed to histidine, accountsfor the largest fraction of rhodopsin mutations in the United States.Several other studies have reported other mutations which also correlatewith the disease. These mutations include Thr58Arg, Pro347Leu,Pro347Ser, as well as deletion of Ile-255. In 2000, a rare mutation incodon 23 was reported causing autosomal dominant retinitis pigmentosa,in which proline changed to alanine However, this study showed that theretinal dystrophy associated with this mutation was characteristicallymild in presentation and course. Furthermore, there was greaterpreservation in electroretinography amplitudes than the more prevalentPro23His mutation.

III. Molecular Diagnostics

While new molecular diagnostic tests are being developed and the rangeof available tests is increasing rapidly, the vast majority of geneticdisorders still lack a genetic test with uncharacterized genetic basis.Certain aspects of the present methods provide tissue or cell-specificanalysis of differences in genetic structure comprising genomicstructure, which may be represented by expression profile between cellsderived from a test subject and a normal cell, for example, a normalcell of the same cell or tissue type.

A genetic test is the analysis of human DNA, RNA, chromosomes, proteinsor certain metabolites in order to detect alterations related to aheritable disorder. This can be accomplished by directly examining theDNA or RNA that makes up a gene (direct testing), looking at markersco-inherited with a disease-causing gene (linkage testing), assayingcertain metabolites (biochemical testing), or examining the chromosomes(cytogenetic testing). Genetic testing is often the best way to confirma diagnosis in a patient with signs or symptoms suggestive of a geneticdisease. The technique chosen depends on both the clinical question andthe predictive value of the available tests.

A. RNA Sequencing

RNA is less stable in the cell, and also more prone to nuclease attackexperimentally. As RNA is generated by transcription from DNA, theinformation is already present in the cell's DNA. However, it issometimes desirable to sequence RNA molecules. In particular, inEukaryotes RNA molecules are not necessarily co-linear with their DNAtemplate, as introns are excised. To sequence RNA, the usual method isfirst to reverse transcribe the sample to generate DNA fragments. Thiscan then be sequenced as DNA sequencing.

Microarray technology and varied multiplex amplification methods haveshown that RNA is valid as a target for routine molecular diagnosticsand for future point-of-care testing. Detection of genetic defects inRNA profile in tissue-specific diseases or disorders may lend moreaccuracy and objectivity and provide more information to the diagnosticprocess than DNA-based detection methods. The main challenge usingprotein as a target for routine diagnostics has been low sensitivity,reproducibility and specificity. However, RNA and not DNA as a targetfor routine diagnostics may give the information of clinical activity,regulation or processes in addition to higher or equal sensitivity,reproducibility and specificity compared to DNA as target. For more than10 years new methods of isolation, purification and stabilization ofmRNA has been developed for routine diagnostics making the RNA very muchsuited as a marker for development of new diagnostics methods and evendrugs.

The four aspects that are relevant to molecular diagnostics or genefunction are levels of RNA expression, tissue specificity of RNAexpression, timing of RNA expression, and the primary sequence of theRNA. By just examining the RNA in the target tissue or cells withcertain aspects of the present invention, it will be much easier toidentify a specific defect than having an entire genomic sequence, asthe functional consequences of changes in DNA control regions are notyet understood, but such differences would impact RNA levels, timing, ortissue distribution.

In certain aspects, RNA sequencing may be used to identify the geneticdefect in the specific cell types derived from iPS cells. RNA-Seq, alsocalled “Whole Transcriptome Shotgun Sequencing” (“WTSS”) and “arevolutionary tool for transcriptomics,” refers to the use ofHigh-throughput sequencing technologies to sequence cDNA in order to getinformation about a sample's RNA content, a technique that is quicklybecoming invaluable in the study of genetic diseases (Denoeud et al.,2008). Thanks to the deep coverage and base level resolution provided bynext-generation sequencing instruments, RNA-Seq provides efficient waysto measure transcriptome data experimentally, and to get informationsuch as how different alleles of a gene are expressed, detectpost-transcriptional mutations or identifying gene fusions.

B. Microarray

A DNA microarray is a multiplex technology used in molecular biology andin medicine. It consists of an arrayed series of thousands ofmicroscopic spots of DNA oligonucleotides, called features, eachcontaining picomoles of a specific DNA sequence. This can be a shortsection of a gene or other DNA element that are used as probes tohybridize a cDNA or cRNA sample (called target) under high-stringencyconditions. Probe-target hybridization is usually detected andquantified by detection of fluorophore-, silver-, orchemiluminescence-labeled targets to determine relative abundance ofnucleic acid sequences in the target.

In standard microarrays, the probes are attached to a solid surface by acovalent bond to a chemical matrix (via epoxy-silane, amino-silane,lysine, polyacrylamide or others). The solid surface can be glass or asilicon chip, in which case they are commonly known as gene chip orcolloquially Affy chip when an Affymetrix chip is used. Other microarrayplatforms, such as Illumina, use microscopic beads, instead of the largesolid support. DNA arrays are different from other types of microarrayonly in that they either measure DNA or use DNA as part of its detectionsystem.

DNA microarrays can be used to measure changes in expression levels, todetect single nucleotide polymorphisms (SNPs), in genotyping or inresequencing mutant genomes for determination of genetic variation incertain aspects. More specifically, DNA microarrays can be used todetect DNA (as in comparative genomic hybridization), or detect RNA(most commonly as cDNA after reverse transcription) that may or may notbe translated into proteins. The process of measuring gene expressionvia cDNA is called expression analysis or expression profiling. Infurther aspects, the genetic defect in the specific cell types derivedfrom iPS cells may be identified by gene expression profiling, themeasurement of the activity (the expression) of thousands of genes atonce, to create a global picture of cellular function. Many experimentsof this sort measure an entire genome simultaneously, that is, everygene present in a particular cell. Examples of gene expression profilinginclude, but are not limited to, DNA microarray, SAGE, or qPCR.

DNA microarray technology measures the relative activity of previouslyidentified target genes. This can be a short section of a gene or otherDNA element that are used as probes to hybridize a cDNA or cRNA sample(called target) under high-stringency conditions. Sequence basedtechniques, like serial analysis of gene expression (SAGE, SuperSAGE)may be also used for gene expression profiling. SuperSAGE is especiallyaccurate and can measure any active gene, not just a predefined set.Real-time polymerase chain reaction, also called quantitative real timepolymerase chain reaction (Q-PCR/qPCR) or kinetic polymerase chainreaction, is a laboratory technique based on the polymerase chainreaction, which is used to amplify and simultaneously quantify atargeted cDNA molecule. It enables both detection and quantification (asabsolute number of copies or relative amount when normalized to cDNAinput or additional normalizing genes) of a specific sequence in a cDNAsample.

C. Linkage Analysis

Regarding families with a genetic disorder in which the mutations cannotbe located, if the gene is cloned or its location in the genome isidentified by genetic mapping, a limited form of genetic testing can beoffered that is based on linkage analysis. This involves the use ofpolymorphic DNA or RNA sequences that are closely linked to or foundwithin the gene to track the mutant sequence through the family. Thetechnique can be used if two or more generations of affected familymembers are available for study and may need extensive analysis of thefamily for informative markers. This approach may be also limited bypotential errors due to genetic recombination or genetic heterogeneity(if more than one gene locus is potentially responsible for the samedisorder in different families). Despite these limitations,linkage-based testing has been very useful for counseling families withgenetic diseases such as Duchenne's muscular dystrophy, hemophilia,spinal muscular atrophy, and many other disorders.

D. Polymorphism identification methods

Measurement of sequence variants, primarily single nucleotidepolymorphisms (SNPs), provides the fundamental units for linking geneticsequence to traits. Most genes harbor multiple sequence variations(e.g., SNPs, repeats, indels) showing a broad range of frequencies andlinkage disequilibrium among them. Most polymorphisms are nonfunctionaland may serve as markers for functional alleles. In addition to usingsingle polymorphisms, associations are also often made with the use ofhaplotypes, blocks of linked polymorphisms, that may demarcatetrait-significant cis-regions of sequence. High-throughput SNPgenotyping methods are now coming online, such as SNPlex, capable ofscreening thousands of SNP in many samples (Wenz, 2004). Such methodshave been used to establish haplotype maps on a genome-wide basis,including genes involved in drug metabolism, at significant markerdensity (Kamatani et al., 2004).

In certain aspects of the present invention, mRNA expression measured bymicroarrays may be combined with genome-wide linkage analysis, takingthe expression level of each gene as the measured phenotype restrictedto specific tissue or cell type. Heritability of gene expressionphenotypes can be explored through familial genotyping and transmissiondisequilibrium testing in nuclear families (Spielman & Ewens, 1996) orpedigree disequilibrium testing in larger pedigrees (Martin et al.,2000). Using target tissues (derived from iPS cells) from familymembers, this type of analysis is capable of distinguishing between cis-and trans-acting genetic factors and shows an abundance of functionalgenomic loci and a preponderance of trans-acting effects, as expected.

An alternative approach involves the analysis of allele specificexpression in a relevant target tissue, which may also be prepared fromiPS cells; each allele experiences its own regulation in the samecellular environment, with the other allele (for autosomal genes)serving as an internal control. As a result, the method controls fortissue conditions, trans-acting factors, and other environmentalinfluences. Thus, SNPs in exonic and untranslated regions of message,can serve as markers for allele expression levels in individualsheterozygous for these markers. Taking the human solute carrier family15 (H+/peptide transporter), member 2 gene (hPepT2) as one example, ithas been recently described a method for allele-specific measurement ofmRNA expression through primer extension incorporation of fluorescentdideoxy-nucleotide terminating probes after RT-PCR amplification(Pinsonneault et al., 2004). Significant differences in the relativeabundance of each allele in mRNA from kidney tissues demonstrated thepresence of functional cisacting factors. The primer extension reactioncan be multiplexed (Bray et al., 2004) so that it will be possible tosearch for functional cis-acting polymorphisms in a large number ofgenes (Yan et al., 2002). Similar results can be achieved throughmethods employed on other platforms (Wojnowski & Brockmoller, 2004)including the use of matrix-assisted laser desorption/ionizationtime-of-flight spectroscopy (MALDI-TOF; Ding et al., 2004) andallele-specific RT-PCR methodologies (Zhang et al., 2004). Thesetechniques may be extended to unprocessed heterogeneous nuclear RNA(hnRNA) if exonic and untranslated markers are unavailable and hnRNA isabundant enough in the target samples (Hirota et al., 2004).

IV. Stem Cells

In certain embodiments of the invention, there are disclosed methods ofusing induced pluripotent stem cells (iPS cells) for determination oftissue-specific genetic variation. Those iPS cells may be made byreprogramming somatic cells and could be identical to embryonic stemcells in various aspects as described below. Understanding of embryonicstem cell characteristics could help select induced pluripotent stemcells. Reprogramming factors known from stem cell reprogramming studiescould be used for these novel methods. It is further contemplated thatthese induced pluripotent stem cells could be potentially used toreplace embryonic stem cells for therapeutics and research applicationsdue to the ethics hurdle to use the latter.

A. Stem Cells

Stem cells are cells found in most, if not all, multi-cellularorganisms. They are characterized by the ability to renew themselvesthrough mitotic cell division and differentiating into a diverse rangeof specialized cell types. The two broad types of mammalian stem cellsare: embryonic stem cells that are found in blastocysts, and adult stemcells that are found in adult tissues. In a developing embryo, stemcells can differentiate into all of the specialized embryonic tissues.In adult organisms, stem cells and progenitor cells act as a repairsystem for the body, replenishing specialized cells, but also maintainthe normal turnover of regenerative organs, such as blood, skin orintestinal tissues.

As stem cells can be grown and transformed into specialized cells withcharacteristics consistent with cells of various tissues such as musclesor nerves through cell culture, their use in medical therapies has beenproposed. In particular, embryonic cell lines, autologous embryonic stemcells generated through therapeutic cloning, and highly plastic adultstem cells from the umbilical cord blood or bone marrow are touted aspromising candidates. Most recently, the reprogramming of adult cellsinto induced pluripotent stem cells has enormous potential for replacingembryonic stem cells.

B. Embryonic Stem Cells

Embryonic stem cell lines (ES cell lines) are cultures of cells derivedfrom the epiblast tissue of the inner cell mass (ICM) of a blastocyst orearlier morula stage embryos. A blastocyst is an early stageembryo—approximately four to five days old in humans and consisting of50-150 cells. ES cells are pluripotent and give rise during developmentto all derivatives of the three primary germ layers: ectoderm, endodermand mesoderm. In other words, they can develop into each of the morethan 200 cell types of the adult body when given sufficient andnecessary stimulation for a specific cell type. They do not contributeto the extra-embryonic membranes or the placenta.

Nearly all research to date has taken place using mouse embryonic stemcells (mES) or human embryonic stem cells (hES). Both have the essentialstem cell characteristics, yet they require very different environmentsin order to maintain an undifferentiated state. Mouse ES cells may begrown on a layer of gelatin and require the presence of LeukemiaInhibitory Factor (LIF). Human ES cells could be grown on a feeder layerof mouse embryonic fibroblasts (MEFs) and often require the presence ofbasic Fibroblast Growth Factor (bFGF or FGF-2). Without optimal cultureconditions or genetic manipulation (Chambers et al., 2003), embryonicstem cells will rapidly differentiate.

A human embryonic stem cell may be also defined by the presence ofseveral transcription factors and cell surface proteins. Thetranscription factors Oct4, Nanog, and Sox2 form the core regulatorynetwork that ensures the suppression of genes that lead todifferentiation and the maintenance of pluripotency (Boyer et al.,2005). The cell surface antigens most commonly used to identify hEScells include the glycolipids SSEA3 and SSEA4 and the keratan sulfateantigens Tra-1-60 and Tra-1-81.

After twenty years of research, there are no approved treatments orhuman trials using embryonic stem cells. ES cells, being pluripotentcells, require specific signals for correct differentiation—if injecteddirectly into the body, ES cells will differentiate into many differenttypes of cells, causing a teratoma. Differentiating ES cells into usablecells while avoiding transplant rejection are just a few of the hurdlesthat embryonic stem cell researchers still face. Many nations currentlyhave moratoria on either ES cell research or the production of new EScell lines. Because of their combined abilities of unlimited expansionand pluripotency, embryonic stem cells remain a theoretically potentialsource for regenerative medicine and tissue replacement after injury ordisease. However, one way to circumvent these issues is to inducepluripotent status in somatic cells by direct reprogramming.

C. Induced pluripotent Stem Cells and Reprogramming Factors

Induced pluripotent stem cells, commonly abbreviated as iPS cells oriPSCs, are a type of pluripotent stem cell artificially derived from anon-pluripotent cell, typically an adult somatic cell. Inducedpluripotent stem cells are believed to be identical to naturalpluripotent stem cells, such as embryonic stem cells in many respects,such as in terms of the expression of certain stem cell genes andproteins, chromatin methylation patterns, doubling time, embryoid bodyformation, teratoma formation, viable chimera formation, and potency anddifferentiability, but the full extent of their relation to naturalpluripotent stem cells is still being assessed.

IPS cells were first produced in 2006 (Takahashi et al., 2006) frommouse cells and in 2007 from human cells (Takahashi et al., 2007; Yu etal, 2007). This has been cited as an important advancement in stem cellresearch, as it may allow researchers to obtain pluripotent stem cells,which are important in research and potentially have therapeutic uses,without the controversial use of embryos.

The generation of iPS cells is crucial on the reprogramming factors usedfor the induction. The following factors or combination thereof could beused in the methods disclosed in the present invention. In certainaspects, nucleic acids encoding Sox and Oct (preferably Oct3/4) will beincluded into the reprogramming vector. For example, one or morereprogramming vectors may comprise expression cassettes encoding Sox2,Oct4, Nanog and optionally Lin28, or expression cassettes encoding Sox2,Oct4, K1f4 and optionally c-Myc, or expression cassettes encoding Sox2,Oct4, and optionally Esrrb, or expression cassettes encoding Sox2, Oct4,Nanog, Lin28, K1f4, c-Myc, and optionally SV40 Large T antigen. Nucleicacids encoding these reprogramming factors may be comprised in the sameexpression cassette, different expression cassettes, the samereprogramming vector, or different reprogramming vectors.

Oct4 and certain members of the Sox gene family (Sox1, Sox2, Sox3, andSox15) have been identified as crucial transcriptional regulatorsinvolved in the induction process whose absence makes inductionimpossible. Additional genes, however, including certain members of theK1f family (K1f1, K1f2, K1f4, and K1f5), the Myc family (c-Myc, L-Myc,and N-Myc), Nanog, and Lin28, have been identified to increase theinduction efficiency.

Oct4 (Pou5f1) is one of the family of octamer (“Oct”) transcriptionfactors, and plays a crucial role in maintaining pluripotency. Theabsence of Oct4 in Oct4⁺ cells, such as blastomeres and embryonic stemcells, leads to spontaneous trophoblast differentiation, and presence ofOct4 thus gives rise to the pluripotency and differentiation potentialof embryonic stem cells. Various other genes in the “Oct” family,including Oct4's close relatives, Oct1 and Oct6, fail to elicitinduction, thus demonstrating the exclusiveness of Oct-4 to theinduction process.

The Sox family of genes is associated with maintaining pluripotencysimilar to Oct4, although it is associated with multipotent andunipotent stem cells in contrast with Oct4, which is exclusivelyexpressed in pluripotent stem cells. While Sox2 was the initial geneused for induction by Takahashi et al. (2006), Wernig et al. (2007), andYu et al. (2007), other genes in the Sox family have been found to workas well in the induction process. Sox1 yields iPS cells with a similarefficiency as Sox2, and genes Sox3, Sox15, and Sox18 also generate iPScells, although with decreased efficiency.

In embryonic stem cells, Nanog, along with Oct4 and Sox2, is necessaryin promoting pluripotency. Therefore, it was surprising when Takahashiet al. (2006) reported that Nanog was unnecessary for induction althoughYu et al. (2007) has reported it is possible to generate iPS cells withNanog as one of the factors.

Lin28 is an mRNA binding protein expressed in embryonic stem cells andembryonic carcinoma cells associated with differentiation andproliferation. Thompson et al. demonstrated it is a factor in iPSgeneration, although it is unnecessary.

K1f4 of the K1f family of genes was initially identified by Takahashi etal. (2006) and confirmed by Wernig et al. (2007) as a factor for thegeneration of mouse iPS cells and was demonstrated by Takahashi et al.(2007) as a factor for generation of human iPS cells. However, Yu et al.(2007) reported that K1f4 was unnecessary for generation of human iPScells and in fact failed to generate human iPS cells. K1f2 and K1f4 werefound to be factors capable of generating iPS cells, and related genesK1f1 and K1f5 did as well, although with reduced efficiency.

The Myc family of genes are proto-oncogenes implicated in cancer.Takahashi et al. (2006) and Wernig et al. (2007) demonstrated that c-Mycis a factor implicated in the generation of mouse iPS cells andTakahashi et al. (2007) demonstrated it was a factor implicated in thegeneration of human iPS cells. However, Yu et al. (2007) and Takahashiet al. (2007) reported that c-Myc was unnecessary for generation ofhuman iPS cells. Usage of the “Myc” family of genes in induction of iPScells is troubling for the eventuality of iPS cells as clinicaltherapies, as 25% of mice transplanted with c-Myc-induced iPS cellsdeveloped lethal teratomas. N-Myc and L-Myc have been identified toinduce in the stead of c-myc with similar efficiency. SV40 large antigenmay be used to reduce or prevent the cytotoxcity which may occur whenc-Myc is expressed.

The reprogramming proteins used in the present invention can besubstituted by protein homologs with about the same reprogrammingfunctions. Nucleic acids encoding those homologs could also be used forreprogramming. Conservative amino acid substitutions are preferred--thatis, for example, aspartic-glutamic as polar acidic amino acids;lysine/arginine/histidine as polar basic amino acids;leucine/isoleucine/methionine/valine/alanine/glycine/proline asnon-polar or hydrophobic amino acids; serine/threonine as polar oruncharged hydrophilic amino acids. Conservative amino acid substitutionalso includes groupings based on side chains. For example, a group ofamino acids having aliphatic side chains is glycine, alanine, valine,leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine. Forexample, it is reasonable to expect that replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the properties of theresulting polypeptide. Whether an amino acid change results in afunctional polypeptide can readily be determined by assaying thespecific activity of the polypeptide.

V. Reprogramming Factors Expression

In certain aspects of the present invention, iPS cells are maded fromreprogramming somatic cells using reprogramming factors. The somaticcell in the present invention may be any somatic cell that can beinduced to pluripotency, such as a fibroblast, a keratinocyte, ahematopoietic cell, a mesenchymal cell, a liver cell, a stomach cell, ora β cell. In a prefered aspect, T cells may be used as source of somaticcells for reprogramming (see U.S. Application No. 61/184,546,incorporated herein by reference).

Reprogramming factors may be expressed from expression cassettescomprised in one or more vectors, such as an integrating vector or anepisomal vector. In a further aspect, reprogramming proteins could beintroduced directly into somatic cells by protein transduction (see U.S.Application No. 61/172,079, incorporated herein by reference).

A. Integrating Vectors

IPS cells may be derived by transfection of certain nucleic acids orgenes encoding reprogramming proteins into non-pluripotent cells, suchas T cells, in the present invention. Transfection is typically achievedthrough integrating viral vectors in the current practice, such asretroviruses. Transfected genes may include the master transcriptionalregulators Oct4 (Pouf51) and Sox2, although it is suggested that othergenes enhance the efficiency of induction. After a critical period,small numbers of transfected cells may begin to become morphologicallyand biochemically similar to pluripotent stem cells, and could beisolated through morphological selection, doubling time, or through areporter gene and antibiotic infection.

In November 2007, a milestone was achieved by creating iPS from adulthuman fibroblasts from two independent research teams' studies (Yu etal., 2007; Takahashi et al., 2007). With the same principle used earlierin mouse models, Takahashi et al. (2007) had successfully transformedhuman fibroblasts into pluripotent stem cells using the same fourpivotal genes: Oct4, Sox2, K1f4, and c-Myc with a retroviral system butc-Myc is oncogenic. Yu et al. (2007) used Oct4, Sox2, NANOG, and adifferent gene LIN28 using a lentiviral system avoiding the use ofc-Myc.

As described above, induction of pluripotent stem cells from humandermal fibroblasts has been achieved using retroviruses or lentiviralvectors for ectopic expression of reprogramming genes. Recombinantretroviruses such as the Moloney murine leukemia virus have the abilityto integrate into the host genome in a stable fashion. They contain areverse transcriptase which allows integration into the host genome.Lentiviruses are a subclass of Retroviruses. They are widely adapted asvectors thanks to their ability to integrate into the genome ofnon-dividing as well as dividing cells. The viral genome in the form ofRNA is reverse-transcribed when the virus enters the cell to produceDNA, which is then inserted into the genome at a random position by theviral integrase enzyme.

B. Episomal vectors

These reprogramming methods may also make use of extra-chromosomallyreplicating vectors (i.e., episomal vectors), which are vectors capableof replicating episomally to make iPS cells essentially free ofexogenous vector or viral elements (see U.S. Application No. 61/058,858,incorporated herein by reference; Yu et al., 2009). A number of DNAviruses, such as adenoviruses, Simian vacuolating virus 40 (SV40) orbovine papilloma virus (BPV), or budding yeast ARS (AutonomouslyReplicating Sequences)-containing plasmids replicate extra-chromosomallyor episomally in mammalian cells. These episomal plasmids areintrinsically free from all these disadvantages (Bode et al., 2001)associated with integrating vectors. For example, a lymphotrophic herpesvirus-based including or Epstein Barr Virus (EBV) as defined above mayreplicate extra-chromosomally and help deliver reprogramming genes tosomatic cells.

For example, the plasmid-based approach used in the invention mayextract robust elements necessary for the successful replication andmaintenance of an EBV element-based system without compromising thesystem's tractability in a clinical setting as described in detailbelow. The essential EBV elements are OriP and EBNA-1 or their variantsor functional equivalents. An additional advantage of this system isthat these exogenous elements will be lost with time after beingintroduced into cells, leading to self-sustained iPS cells essentiallyfree of exogenous elements.

The use of plasmid- or liposome-based extra-chromosomal vectors, e.g.,oriP-based vectors, and/or vectors encoding a derivative of EBNA-1permit large fragments of DNA to be introduced to a cell and maintainedextra-chromosomally, replicated once per cell cycle, partitioned todaughter cells efficiently, and elicit substantially no immune response.In particular, EBNA-1, the only viral protein required for thereplication of the oriP-based expression vector, does not elicit acellular immune response because it has developed an efficient mechanismto bypass the processing required for presentation of its antigens onMHC class I molecules (Levitskaya et al., 1997). Further, EBNA-1 can actin trans to enhance expression of the cloned gene, inducing expressionof a cloned gene up to 100-fold in some cell lines (Langle-Rouault etal., 1998; Evans et al., 1997). Finally, the manufacture of suchoriP-based expression vectors is inexpensive.

Other extra-chromosomal vectors include other lymphotrophic herpesvirus-based vectors. Lymphotrophic herpes virus is a herpes virus thatreplicates in a lymphoblast (e.g., a human B lymphoblast) and becomes aplasmid for a part of its natural life-cycle. Herpes simplex virus (HSV)is not a “lymphotrophic” herpes virus. Exemplary lymphotrophic herpesviruses include, but are not limited to EBV, Kaposi's sarcoma herpesvirus (KSHV); Herpes virus saimiri (HS) and Marek's disease virus (MDV).Also other sources of episome-base vectors are contemplated, such asyeast ARS, adenovirus, SV40, or BPV.

To circumvent potential problems from viral gene delivery, two groupsthis year reported on a collaboration that has succeeded intransposon-based approaches for producing pluripotency in human cellswithout using viral vectors (Woltjen et al., 2009; Kaji et al., 2009).Stable iPS cells were produced in both human and mouse fibroblasts usingvirus-derived 2A peptide sequences to create a multicistronic vectorincorporating the reprogramming factors, delivered to the cell by thepiggyBac transposon vector. The 2A-linked reprogramming factors, notrequired in the established iPS cell lines, were then removed. Thesestrategies could be similarly applied to reprogram T cell in certainaspects of the present invention.

C. Protein Transduction

One possible way to avoid introducing exogenous genetic modifications totarget cells would be to deliver the reprogramming proteins directlyinto cells, rather than relying on the transcription from deliveredgenes. Previous studies have demonstrated that various proteins can bedelivered into cells in vitro and in vivo by conjugating them with ashort peptide that mediates protein transduction, such as HIV tat andpoly-arginine. A recent study demonstrated that murine fibroblasts canbe fully reprogrammed into pluripotent stem cells by direct delivery ofrecombinant reprogramming proteins (Zhou et al., 2009). More details ofthe methods for reprogramming cells with protein transduction have beendisclosed in U.S. Application No. 61/172,079 incorporated herein byreference.

In certain aspects of the present invention, protein transductiondomains could been used to introduce reprogramming proteins directlyinto T cells. Protein transduction could be a method for enhancing thedelivery of reprogramming proteins into cells. For example, a region ofthe TAT protein which is derived from the HIV Tat protein can be fusedto a target protein allowing the entry of the target protein into thecell. The advantages of using fusions of these transduction domains isthat protein entry is rapid, concentration-dependent and appears to workwith different cell types.

In a further aspect of the present invention, nuclear localizationsequence may also be used to facilitate nuclear entry of reprogrammingproteins. Nuclear localization signals (NLS) have been described forvarious proteins. The mechanism of protein transport to the nucleus isthrough the binding of a target protein containing a nuclearlocalization signal to alpha subunit of karyopherin. This is followed bytransport of the target protein:karyopherin complex through the nuclearpore and into the nucleus. However, reprogramming proteins are oftentranscription factors which may have endogenous nuclear localizationsequences. Therefore, nuclear localization sequences may not benecessary.

The direct introduction of reprogramming proteins into somatic cells maybe used in the present invention, with reprogramming proteinsoperatively linked to a protein transduction domain (PTD), either bycreating a fusion protein comprising such a domain or by chemicallycross-linking the reprogramming protein and PTD via functional groups oneach molecule.

Standard recombinant nucleic acid methods can be used to express one ormore transducible reprogramming proteins used herein. In one embodiment,a nucleic acid sequence encoding the transducible protein is cloned intoa nucleic acid expression vector, e.g., with appropriate signal andprocessing sequences and regulatory sequences for transcription andtranslation. In another embodiment, the protein can be synthesized usingautomated organic synthetic methods.

In addition, there have been several methods that may also help thetransport of proteins into cells, one ore more of which can be usedalone or in combination with the methods using the protein transductiondomains, including, but not limited to, microinjection, electroporation,and the use of liposomes. Most of these methods may need a purifiedpreparation of protein. Purification of recombinant proteins is oftenfacilitated by the incorporation of an affinity tag into the expressionconstruct, making the purification step fast and efficient.

VI. IPS Cell Selection, Culturing, and Differentiation

In certain aspects of the invention, after one or more reprogrammingfactors are introduced into somatic cells of a test subject, cells willbe cultured for expansion (optionally selected for the presence ofvector elements like positive selection or screenable marker toconcentrate transfected cells). Reprogramming vectors may expressreprogramming factors in these cells and replicate and partition alongwith cell division. Alternatively, reprogramming proteins could enterthese cells and their progeny by replenishing medium containing thereprogramming proteins. These reprogramming factors will reprogramsomatic cell genome to establish a self-sustaining pluripotent state,and in the meantime or after removal of positive selection of thepresence of vectors, exogenous genetic elements will be lost gradually,or there is no need to add reprogramming proteins.

These induced pluripotent stem cells could be selected from progenycells based on embryonic stem cell characteristics because they areexpected to be substantially identical to pluripotent embryonic stemcells. An additional negative selection step could be also employed toaccelerate or help selection of iPS cells essentially free of exogenousgenetic elements by testing the absence of reprogramming vector DNA orusing selection markers, such as reporters.

After iPS cells are selected or isolated, differentiation into specificcells types may be induced from iPS cells for determination oftissue-specific genetic variation. The specific cell types may becomprised in a selected tissue, such as retina.

A. IPS Cell Selection

The successfully generated iPSCs from previous studies were remarkablysimilar to naturally-isolated pluripotent stem cells (such as mouse andhuman embryonic stem cells, mESCs and hESCs, respectively) in thefollowing respects, thus confirming the identity, authenticity, andpluripotency of iPSCs to naturally-isolated pluripotent stem cells.Thus, induced pluripotent stem cells generated from the methodsdisclosed in this invention could be selected based on one or more offollowing embryonic stem cell characteristics.

i. Cellular Biological Properties

Morphology: iPSCs are morphologically similar to ESCs. Each cell mayhave round shape, dual nucleoli or large nucleolus and scant cytoplasm.Colonies of iPSCs could be also similar to that of ESCs. Human iPSCsform sharp-edged, flat, tightly-packed colonies similar to hESCs andmouse iPSCs form the colonies similar to mESCs, less flatt and moreaggregated colonies than that of hESCs.

Growth properties: Doubling time and mitotic activity are cornerstonesof ESCs, as stem cells must self-renew as part of their definition.iPSCs could be mitotically active, actively self-renewing,proliferating, and dividing at a rate equal to ESCs.

Stem Cell Markers: iPSCs may express cell surface antigenic markersexpressed on ESCs. Human iPSCs expressed the markers specific to hESC,including, but not limited to, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81,TRA-2-49/6E, and Nanog. Mouse iPSCs expressed SSEA-1 but not SSEA-3 norSSEA-4, similarly to mESCs.

Stem Cell Genes: iPSCs may express genes expressed in undifferentiatedESCs, including Oct4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4,and hTERT.

Telomerase Activity: Telomerases are necessary to sustain cell divisionunrestricted by the Hayflick limit of ˜50 cell divisions. hESCs expresshigh telomerase activity to sustain self-renewal and proliferation, andiPSCs also demonstrate high telomerase activity and express hTERT (humantelomerase reverse transcriptase), a necessary component in thetelomerase protein complex.

Pluripotency: iPSCs will be capable of differentiation in a fashionsimilar to ESCs into fully differentiated tissues.

Neural Differentiation: iPSCs could be differentiated into neurons,expressing βIII-tubulin, tyrosine hydroxylase, AADC, DAT, ChAT, LMX1B,and MAP2. The presence of catecholamine-associated enzymes may indicatethat iPSCs, like hESCs, may be differentiable into dopaminergic neurons.Stem cell-associated genes will be downregulated after differentiation.

Cardiac Differentiation: iPSCs could be differentiated intocardiomyocytes that spontaneously begin beating. Cardiomyocytes expresscTnT, MEF2C, MYL2A, MYHCβ, and NKX2.5. Stem cell-associated genes willbe downregulated after differentiation.

Teratoma Formation: iPSCs injected into immunodeficient mice mayspontaneously form teratomas after certain time, such as nine weeks.Teratomas are tumors of multiple lineages containing tissue derived fromthe three germ layers endoderm, mesoderm and ectoderm; this is unlikeother tumors, which typically are of only one cell type. Teratomaformation is a landmark test for pluripotency.

Embryoid Body: hESCs in culture spontaneously form ball-like embryo-likestructures termed “embryoid bodies,” which consist of a core ofmitotically active and differentiating hESCs and a periphery of fullydifferentiated cells from all three germ layers. iPSCs may also formembryoid bodies and have peripheral differentiated cells.

Blastocyst Injection: hESCs naturally reside within the inner cell mass(embryoblast) of blastocysts, and in the embryoblast, differentiate intothe embryo while the blastocyst's shell (trophoblast) differentiatesinto extraembryonic tissues. The hollow trophoblast is unable to form aliving embryo, and thus it is necessary for the embryonic stem cellswithin the embryoblast to differentiate and form the embryo. iPSCsinjected by micropipette into a trophoblast to generate a blastocysttransferred to recipient females, may result in chimeric living mousepups: mice with iPSC derivatives incorporated all across their bodieswith 10%-90 and chimerism.

ii. Epigenetic reprogramming

Promoter Demethylation: Methylation is the transfer of a methyl group toa DNA base, typically the transfer of a methyl group to a cytosinemolecule in a CpG site (adjacent cytosine/guanine sequence). Widespreadmethylation of a gene interferes with expression by preventing theactivity of expression proteins or recruiting enzymes that interferewith expression. Thus, methylation of a gene effectively silences it bypreventing transcription. Promoters of pluripotency-associated genes,including Oct4, Rex1, and Nanog, may be demethylated in iPSCs, showingtheir promoter activity and the active promotion and expression ofpluripotency-associated genes in iPSCs.

Histone Demethylation: Histones are compacting proteins that arestructurally localized to DNA sequences that can effect their activitythrough various chromatin-related modifications. H3 histones associatedwith Oct/4, Sox2, and Nanog may be demethylated to activate theexpression of Oct4, Sox2, and Nanog.

B. Culturing of iPS cells

After somatic cells are introduced with reprogramming factors using thedisclosed methods, these cells may be cultured in a medium sufficient tomaintain the pluripotency. Culturing of induced pluripotent stem (iPS)cells generated in this invention can use various medium and techniquesdeveloped to culture primate pluripotent stem cells, more specially,embryonic stem cells, as described in U.S. Pat. App. 20070238170 andU.S. Pat. App. 20030211603. It is appreciated that additional methodsfor the culture and maintenance of human pluripotent stem cells, aswould be known to one of skill, may be used with the present invention.

In certain embodiments, undefined conditions may be used; for example,pluripotent cells may be cultured on fibroblast feeder cells or a mediumwhich has been exposed to fibroblast feeder cells in order to maintainthe stem cells in an undifferentiated state. Alternately, pluripotentcells may be cultured and maintained in an essentially undifferentiatedstate using defined, feeder-independent culture system, such as a TeSRmedium (Ludwig et al., 2006a; Ludwig et al., 2006b). Feeder-independentculture systems and media may be used to culture and maintainpluripotent cells. These approaches allow human embryonic stem cells toremain in an essentially undifferentiated state without the need formouse fibroblast “feeder layers.” As described herein, variousmodifications may be made to these methods in order to reduce costs asdesired.

For example, like human embryonic stem (hES) cells, iPS cells can bemaintained in 80% DMEM (Gibco #10829-018 or #11965-092), 20% definedfetal bovine serum (FBS) not heat inactivated (or human AB serum), 1%non-essential amino acids, 1 mM L-glutamine, and 0.1 mMβ-mercaptoethanol. Alternatively, iPS cells can be maintained inserum-free medium, made with 80% Knock-Out DMEM (Gibco #10829-018), 20%serum replacement (Gibco #10828-028), 1% non-essential amino acids, 1 mML-glutamine, and 0.1 mM β-mercaptoethanol. Just before use, human bFGFmay be added to a final concentration of about 4 ng/mL (WO 99/20741) orzebrafish bFGF may be used instead as in the Examples.

Various matrix components may be used in culturing and maintaining humanpluripotent stem cells. For example, collagen IV, fibronectin, laminin,and vitronectin in combination may be used to coat a culturing surfaceas a means of providing a solid support for pluripotent cell growth, asdescribed in Ludwig et al. (2006a; 2006b), which are incorporated byreference in its entirety.

Matrigel™ may also be used to provide a substrate for cell culture andmaintenance of human pluripotent stem cells. Matrigel™ is a gelatinousprotein mixture secreted by mouse tumor cells and is commerciallyavailable from BD Biosciences (New Jersey, USA). This mixture resemblesthe complex extracellular environment found in many tissues and is usedby cell biologists as a substrate for cell culture.

IPS cells, like ES cells, have characteristic antigens that can beidentified or confirmed by immunohistochemistry or flow cytometry, usingantibodies for SSEA-1, SSEA-3 and SSEA-4 (Developmental StudiesHybridoma Bank, National Institute of Child Health and HumanDevelopment, Bethesda Md.), and TRA-1-60 and TRA-1-81 (Andrews et al.,1987). Pluripotency of embryonic stem cells can be confirmed byinjecting approximately 0.5-10×10⁶ cells into the rear leg muscles of8-12 week old male SCID mice. Teratomas develop that demonstrate atleast one cell type of each of the three germ layers.

C. Differentiation of iPS Cells

Various approaches may be used with the present invention todifferentiate genetically iPS cells into specific cell lineagesincluding, but not limited to, retina epithelium cells, neural retinacells, hematopoietic cells, myocytes (e.g., cardiomyocytes), neurons,fibroblasts and epidermal cells, and tissues or organs derivedtherefrom. Differentiation into retina cells may be an example.

The retinal pigment epithelium (RPE) is the pigmented cell layer justoutside the neurosensory retina that nourishes retinal visual cells, andis firmly attached to the underlying choroid and overlying retinalvisual cells. The RPE is composed of a single layer of hexagonal cellsthat are densely packed with pigment granules. The retinal pigmentepithelium is involved in the phagocytosis of the outer segment ofphotoreceptor cells and it is also involved in the vitamin A cycle whereit isomerizes all trans retinol to 11-cis retinal. The retinal pigmentepithelium also serves as the limiting transport factor that maintainsthe retinal environment by supplying small molecules such as amino acid,ascorbic acid and D-glucose while remaining a tight barrier to choroidalblood borne substances. Homeostasis of the ionic environment ismaintained by a delicate transport exchange system. When viewed from theouter surface, these cells are smooth and hexagonal in shape. When seenin section, each cell consists of an outer non-pigmented part containinga large oval nucleus and an inner pigmented portion which extends as aseries of straight thread-like processes between the rods, this beingespecially the case when the eye is exposed to light.

The isolation of RPE cells from human patient may be technicallychallenging and complicated. In certain aspects of the presentinvention, RPE cells may be obtained by differentiation of iPS cells forgenetic defect identification. For example, RPE cells can bedifferentiated by methods disclosed in WO 2008/129554, Osakada et al.(2008), Osakada et al. (2009), and Hirami et al. (2009). For example,human iPS cells could be dissociated into small clumps of cells andseeded in Petri dishes as suspension cultures with a serum-free medium(SFEB/DL) containing Wnt and Nodal antagonists. Under these conditions,iPS cells could form embryoid body-like aggregates. On day 20,aggregates may be plated onto glass slides coated with poly-D-lysine,laminin, and fibronectin for further RPE differentiation.

The iPS cells may also be differentiated into cardiac or blood cells incertain aspects. Exemplary methods of cardiac differentiation of iPScells may include embryoid body (EB) methods (Zhang, et al., 2009), orOP9 stroma cell methods (Narazaki, et al., 2008), or growthfactor/chemical methods (see U.S. Patent Publn. 20080038820,20080226558, 20080254003 and 20090047739, all incorporated herein byreference in their entirety). Exemplary methods of hematopoieticdifferentiation of iPS cells may include, but are not limited to,methods disclosed by U.S. Application No. 61/088,054 and No. 61/156,304,both incorporated herein by reference in their entirety, or embryoidbody (EB) based methods (Chadwick et al., 2003; Ng et al., 2005).Fibronectin differentiation methods may also be used for blood lineagedifferentiation, as exemplified in Wang et al., 2007.

VII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1

IPS cells are made from a patient diagnosed with retinitis pigmentosa.As a control, iPS cells are also made from the patient's family member,preferably a sibling, who is without retinitis pigmentosa. Human iPScell lines are obtained by the lentiviral-mediated transduction of fourtranscription factors (OCT4, SOX2, NANOG and LIN28) as previouslydescribed (Yu et al., 2007). The iPS cells derived both from the patientand the sibling are maintained on matrigel™ and cultured in TeSR medium.

A single cell suspension is made by incubating colonies in TrypLe, arecombinant trypsin-like enzyme (Invitrogen, Carlsbad, Calif.) at 37° C.for 7 minutes, washed twice with TeSR medium containing the apoptosisinhibitor H1152 and soybean trypsin inhibitor, and resuspended in 0.5 mlof cold PBS containing H1152 and soybean trypsin inhibitor. Thesuspension cultures are seeded on a gelatin-coated dish with serum-freemedium supplemented with 100 ng/ml Dkk-1, a Wnt antagonist and 500 ng/mlLefty A, a Nodal antagonist for 18-20 days to form aggregates. Theaggregates are then plated onto culture slides coated withpoly-D-lysine, laminin, and fibronectin and incubated in differentiationmedium (G-MEM (GIBCO) containing 10% KSR (Knock-out serum replacement),0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM2-mercaptoethanol, 50 units/ml penicillin, and 50 μg/ml streptomycin).RPE cells are selected and enriched by polygonal morphology,pigmentation and molecular markers like RPE-65 and ZO-1, a tightjunction marker.

Total RNA is extracted from the RPE cells using TRIzol reagent(Invitrogen) or TRI-Reagent (Sigma). cDNA synthesis is carried out usingMoloney murine leukemia virus reverse transcriptase (M-MLV RT) andrandom primers, according to the manufacturer's instructions (PromegaCorporation, Madison, Wis.). Polymerase chain reaction (PCR) is carriedout using standard protocols with Taq DNA Polymerase (Gibco-BRL). Q-PCR,cDNA sequencing and microarray are used to compare the RPE cells derivedfrom the patient and the related normal family member to determine oneor more genetic variation.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for determining the presence of a tissue-specific geneticvariation in a test subject relative to a selected control geneticstructure, comprising: a) obtaining genetic material of a differentiatedcell of a selected tissue, the cell having been prepared bydifferentiating an induced pluripotent stem (iPS) cell obtained byreprogramming a somatic cell of the subject; and b) testing the geneticmaterial of said differentiated cell to compare one or more geneticstructure of said genetic material with one or more control geneticstructures to determine the presence of such a genetic variation incells of the tissue of the subject.
 2. The method of claim 1, whereinthe subject has or is suspected of having a genetic abnormality orgenetic disease.
 3. The method of claim 1, wherein said selected tissueis retina.
 4. The method of claim 3, wherein said differentiated cell isa neural retina cell.
 5. The method of claim 3, wherein saiddifferentiated cell is a retina pigment epithelium (RPE) cell.
 6. Themethod of claim 1, wherein said testing comprises RNA or DNA sequencing.7. The method of claim 1, wherein said testing employs microarrayanalysis.
 8. The method of claim 1, wherein said genetic materialcomprises RNA or DNA.
 9. The method of claim 1, wherein said geneticstructure is a nucleotide sequence.
 10. The method of claim 1, whereinsaid genetic structure is represented by an expression profile on RNAlevel or protein level.
 11. The method of claim 1, wherein said geneticvariation is a polymorphism.
 12. The method of claim 11, wherein saidgenetic variation is a single nucleotide polymorphism variation.
 13. Themethod of claim 1, wherein said genetic variation is a gene mutation.14. The method of claim 1, wherein said genetic variation is representedby differential expression on RNA level or protein level.
 15. The methodof claim 1, wherein said genetic variation is variation of one or morecoding sequences.
 16. The method of claim 1, wherein said geneticvariation is variation of one or more gene regulatory elements.
 17. Themethod of claim 16, wherein said gene regulatory element is a promoter,enhancer, silencer or responsive element.
 18. The method of claim 16,wherein said gene regulatory element is microRNA.
 19. The method ofclaim 1, wherein said genetic variation is epigenetic variation.
 20. Themethod of claim 19, wherein said genetic variation is variation inmethylation of the genetic structure.
 21. The method of claim 1, whereinthe control genetic structure is one known not to have a selectedgenetic variation.
 22. The method of claim 21, wherein the controlgenetic structure is comprised in genetic material from a normal tissue.23. The method of claim 1, wherein the control genetic structure iscomprised in genetic material, wherein the genetic material is from aselected normal cell prepared by differentiating an iPS cell obtained byreprogramming a normal somatic cell of a control subject.
 24. The methodof claim 23, wherein the selected normal cell is of the same tissue typeas the selected tissue of the test subject.
 25. The method of claim 1,wherein the control genetic structure is of a control subject being arelative of the test subject.
 26. The method of claim 25, wherein thecontrol subject is a sibling of the test subject.
 27. The method ofclaim 22, wherein the normal tissue is of the same tissue type as theselected tissue of the test subject.